The present invention is directed to a method of determining the concentration of antibiotics in bone.
The use of antibiotics for bone diseases is not extensively studied due to insufficient information on their disposition and relationship between bone concentration and pharmacological effects [Stepensky, D., et al., Clinical Pharmokinetics (2003), 42:863-881, p. 865, p 875].
The pharmacologic management of bone infections is difficult. Systemic antibiotic therapy alone does not usually eradicate bacteria because of poor penetration into bone. Adverse effects are increased when high doses of antibiotics are administered over long durations of treatment. Confounding this issue is the increasing prevalence of highly resistant pathogens [Winkler, H., et al., J. Antimicrob. Chemotherapy (2000), 46:423-428, p 423]. Tigecycline is currently indicated for susceptible pathogens isolated from complicated skin and skin structure infections and complicated intra-abdominal infections. Tigecycline is widely distributed and effectively penetrates bone. It is highly effective on resistant organisms. An expanded indication for treating localized infections in bone tissue could be explored if accurate assay methods for determining antibiotic concentrations in bone were available.
One study [Smilack J. D., et al., Antimicrobial Agents and Chemotherapy (1976), 9:169-171, p 169] reported the measurement of antimicrobial agents in human bone (from hip or knee replacement surgery) using a microbiological disk diffusion method. In this microbiology method, the antibiotics were extracted from pulverized bone with a neutral buffer solution (pH 6.8). The buffer solution was then incubated with designated Bacillus subtilis (or other type) seeded antibiotic medium, the diameter of diffusion of the inhibition zone in an agar plate was measured and quantified with the corresponding antibiotic standard curve. This microbiology assay has a detection limit of <1 to 5 microgram per ml in serum and <0.5 to 3.6 microgram per gram of bone. This assay detected the antibiotic concentration in the majority of the serum samples, however, many corresponding bone samples had no antibiotic concentration detected. Several other studies have reported the use of various acids, such as hydrochloric acid [Elliston, J. T., et al., J. Radioanal. Nuclear Chem. (2005), 263:301-306][Demirbas, A., et al., Resources Conserving and Recycling (1999), 26:251-258, p 252][Christgau, M., et al., J. Periodontal Research (1998), 33:138-149, p. 138], nitric acid and hydrochloric acid mixtures [Roberts, N. B., et al., J. Analytical Atomic Spectrometry (1996), 11:133-138, p. 133], and perchloric acid [Zakrzewska, H., et al., Archives of Oral Biology (2005), 50:309-316, p. 309] to dissolve animal bone, human bone, or tooth. In these methods, inorganic ions such as fluoride, phosphate, calcium and other trace metal ions were measured with their respective ion-selective electrodes or atomic adsorption methods without instability of the analyte issue. Using these strong acids to dissolve rat or human bone, in preparation for a tigecycline antibiotic bone assay (ABA), would cause instability of the drug and result in difficulties in drug quantification by LC/MS/MS.
Li, et al. developed an ion-paired high-performance liquid chromatography-UV method to determine tigecycline concentration in human polymorphonuclear neutrophils and human serum [Li, C. H., et al. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. (2004), 811:225-229]. This method employed a 5% trichloroacetic acid in Hank's balanced salts solution to lyse cells and precipitate proteins. The drug remaining in the supernatant was assayed using ion-pair chromatography with UV detection. This type of extraction method may not be adapted to a bone assay due to the insolubility of bone in the 5% trichloroacetic acid extraction solvent and the limitation of the UV method.
There remains a need for an improved method for determining the concentration of antibiotics in bone, in order to facilitate the expanded use of antibiotics in bone diseases and infections.